We have devised site-specific DNA recombination strategies to easily move defined segments of DNA into and out of the mouse genome in order to understand the developmental control of gene expression. In previous work we established that the Cre recombinase of phage P1 can efficiently excise a loxP delimited DNA fragment from the genome of a wide variety of eukaryotes, including mice, and that Cre can also direct integration of loxP-tagged exogenous DNA to a loxP site previously placed into the genome. We have placed recombination substrates at defined chromosomal positions using homologous recombination in embryonic stem (ES) cells. Following Cre-mediated targeting of defined promoter-reporter constructs in these cell lines, mice are being generated that will allow dissection of the regional and temporal specific determinants of gene expression in the proenkephalin promoter. Mechanistic analysis of Cre function should provide an increased ability to direct Cre-meditated events in cultured cells and transgenic mice. We have used mutationally altered lox sites to develop a new Cre-mediated targeting strategy that is 20-100 times more efficient than our previously described strategy in a model cultured cell system. This system is now being adapted to the generation of transgenic mice. This is the last year of this project.